Molecules 2014, 19, 20325-20339; doi:10.3390/molecules191220325

molecules ISSN 1420-3049

www.mdpi.com/journal/molecules

Article

Formulation, Characterization, and in Vivo Evaluation of Celecoxib-PVP Solid Dispersion Nanoparticles Using Supercritical Antisolvent Process

Eun-Sol Ha 1,†, Gwang-Ho Choo 1,†, In-Hwan Baek 2 and Min-Soo Kim 1,*

1 College of Pharmacy, Pusan National University, Busan 609-735, Korea;

E-Mails: edel@pusan.ac.kr (E.-S.H.); jimasd@pusan.ac.kr (G.-H.C.) 2 College of Pharmacy, Kyungsung University, Busan 608-736, Korea; E-Mail: baek@ks.ac.kr

† These authors contributed equally to this work.

* Author to whom correspondence should be addressed; E-Mail: minsookim@pusan.ac.kr;

Tel.: +82-51-510-2813; Fax: +82-51-513-6754.

External Editor: Derek J. McPhee

Received: 13 November 2014; in revised form: 1 December 2014 / Accepted: 3 December 2014 /

Published: 4 December 2014

Abstract: The aim of this study was to develop celecoxib-polyvinylpyrrolidone (PVP)

solid dispersion nanoparticles with and without surfactant using the supercritical

antisolvent (SAS) process. The effect of different surfactants such as gelucire 44/14,

poloxamer 188, poloxamer 407, Ryoto sugar ester L1695, and D-α-tocopheryl polyethylene

glycol 1000 succinate (TPGS) on nanoparticle formation and dissolution as well as oral

absorption of celecoxib-PVP K30 solid dispersion nanoparticles was investigated.

Spherical celecoxib solid dispersion nanoparticles less than 300 nm in size were

successfully developed using the SAS process. Analysis by differential scanning

calorimetry and powder X-ray diffraction showed that celecoxib existed in the amorphous

form within the solid dispersion nanoparticles fabricated using the SAS process. The

celecoxib-PVP-TPGS solid dispersion nanoparticles significantly enhanced in vitro

dissolution and oral absorption of celecoxib relative to that of the unprocessed form. The

area under the concentration-time curve (AUC0→24 h) and peak plasma concentration (Cmax)

increased 4.6 and 5.7 times, respectively, with the celecoxib-PVP-TPGS formulation. In

addition, in vitro dissolution efficiency was well correlated with in vivo pharmacokinetic

parameters. The present study demonstrated that formulation of celecoxib-PVP-TPGS

OPEN ACCESS

Molecules 2014, 19 20326

solid dispersion nanoparticles using the SAS process is a highly effective strategy for

enhancing the bioavailability of poorly water-soluble celecoxib.

Keywords: solid dispersion; bioavailability; celecoxib; nanoparticles; supercritical

antisolvent

1. Introduction

Celecoxib, 4-[5-(4-methylphenyl)-3-(trifluoromethyl)pyrazol-1-yl]benzenesulfonamide, is a poorly

water-soluble drug belonging to the class of selective cyclooxygenase-2 (COX-2) inhibitors and is

clinically used in the treatment of acute pain, rheumatoid arthritis, and osteoarthritis [1]. Celecoxib

with an acid dissociation constant (pKa) of 11.1 belongs to the biopharmaceutics classification system

(BSC) class II drug category because of its low solubility and high permeability [2]. Various

formulations of celecoxib such as self-microemulsifying drug delivery systems (SMEDDS),

microparticles, nanoparticles, silica-lipid hybrid microcapsules, and surface solid dispersions have

been studied to evaluate their potential to enhance dissolution of celecoxib [3–8].

Solid dispersion is well established as a formulation system for enhancing the bioavailability of

poorly water-soluble active pharmaceutical ingredients (APIs). Generally, solid dispersions consist of

an API and a hydrophilic polymer such as hydroxypropyl methylcellulose (HPMC),

polyvinylpyrrolidone (PVP), or polyethylene glycol (PEG) [9–11]. Most poorly water-soluble APIs

exist in an amorphous form within the solid dispersion, thereby enhancing their dissolution and oral

absorption by attaining a highly supersaturated state above their equilibrium solubility. Furthermore,

ternary solid dispersions consisting of API, polymer, and surfactant can further enhance dissolution

and in vivo performance of APIs compared to binary solid dispersions [12]. Our group recently

reported that of the 71 combination formulations we evaluated, the most efficient ternary solid

dispersion for enhanced bioavailability of sirolimus was the HPMC/d- α-tocopheryl polyethylene

glycol 1000 succinate (TPGS) followed by the HPMC/Sucroester 15 [13]. Solid dispersions containing

polymer and/or surfactant can be manufactured on the principle of solvent evaporation, melting, and/or

solvent-mediated melting. It has been reported that solid dispersion nanoparticles can be manufactured

using supercritical fluid technology. Supercritical carbon dioxide (Pc = 7.38 MPa, Tc = 31.1 °C) is

widely used as a solvent or antisolvent in the field of nanoparticle formation because it is non-toxic

and non-flammable. Supercritical carbon dioxide can therefore be considered a green solvent

alternative to potential toxic organic solvents in the pharmaceutical industry. The nanoparticle

manufacturing process using supercritical carbon dioxide was developed based on the role of the

supercritical carbon dioxide in the process as either a solvent (rapid expansion of supercritical

solutions) or an antisolvent (supercritical antisolvent process; SAS). Yasuji et al. reviewed particle

design of poorly water-soluble APIs using supercritical fluid technologies [14]. Solid dispersion

nanoparticles manufactured with hydrophilic polymers and surfactants using the SAS process

significantly improved the solubility, dissolution, and oral bioavailability of poorly water-soluble APIs

such as atorvastatin calcium, azithromycin, dutasteride, lercanidipine, nilotinib, valsartan, tadalafil and

telmisartan [15–23].

Molecules 2014, 19 20327

The aim of this study was to develop celecoxib-PVP solid dispersion nanoparticles with and without

surfactant using the SAS process and to evaluate their potential to enhance the dissolution and oral

bioavailability of celecoxib. Recently, Abu-Diak et al. reported that the recrystallization of celecoxib

from supersaturated solution was significantly inhibited by PVP in a concentration-dependent

manner [24]. Therefore, we used PVP K30 as a hydrophilic polymer in this study. The effect of

different surfactants including gelucire 44/14, poloxamer 188, poloxamer 407, Ryoto sugar ester

L1695, and TPGS on the nanoparticle formation, dissolution, and oral absorption of celecoxib-PVP

K30 solid dispersion nanoparticles was investigated. An in vitro-in vivo correlation (IVIVC) study was

also conducted using the in vitro dissolution data and in vivo pharmacokinetic parameters.

2. Results and Discussion

In this study, celecoxib-PVP K30 solid dispersion nanoparticles were formulated using the SAS

process with the aim of enhancing the dissolution and oral absorption of celecoxib. Figure 1 shows the

SEM images of the solid dispersion nanoparticles and Table 1 summarizes the mean particle size and

specific surface area of solid dispersion nanoparticles. All celecoxib-PVP K30 solid dispersion

nanoparticles had regular spherical shape with particle size range of 150–158 nm and specific surface

area of 78–81 m2/g, indicating there was no significant difference between the celecoxib-PVP K30

solid dispersion nanoparticle formulations. The ratio of celecoxib/PVP K30 also did not appear to

influence the morphology and particle size of solid dispersion nanoparticles prepared by the SAS

process. However, the mean particle size and specific surface area of solid dispersion nanoparticles

were significantly affected by addition of the surfactant (Figure 2).

In particular, the particles of the celecoxib-PVP K30-gelucire 44/14 solid dispersion nanoparticles

had a regular spherical shape with mean particle size of 610.3 nm and specific surface area of

21.2 m2/g. As shown in Table 1, the mean particle size and specific surface area of solid dispersion

nanoparticles increased and decreased, respectively, following addition of the surfactant. The increased

mean particle size and aggregation of solid dispersion nanoparticles might be due to the fusion of the

surfactant, resulting in lower melting temperature. The melting points of all surfactants tested were

below 56 °C as stated in the material and methods section. Similar results were previously

reported [15–18]. Nevertheless, solid dispersion nanoparticles with surfactants (with the exception of

gelucire 44/14) showed mean particle sizes below 300 nm.

The crystal state of celecoxib within the solid dispersion nanoparticles was determined by DSC

curves and PXRD patterns and is shown in Figure 3. Raw celecoxib showed a sharp endothermic peak

at about 163 °C. In addition, characteristic diffraction patterns indicating crystalline celecoxib were

observed at 2θ values of 5.31°, 10.68°, 13.00°, 14.83°, 16.08°, 19.63°, 21.49°, 22.13°, 25.36°, and

29.48°. However, the melting peak and diffraction patterns of crystalline celecoxib were not observed

for all solid dispersion nanoparticle formulations. In fact, celecoxib exists in the amorphous form in

solid dispersion nanoparticle fabricated by the SAS process.

Molecules 2014, 19 20328

Figure 1. Scanning electron micrographs of celecoxib-polyvinylpyrrolidone

(celecoxib-PVP) K30 solid dispersion nanoparticles prepared using the supercritical

antisolvent (SAS) process. (A) celecoxib-PVP K30 (2:8); (B) celecoxib-PVP K30-gelucire

44/14; (C) celecoxib-PVP K30-poloxamer 188; (D) celecoxib-PVP K30-poloxamer 407;

(E) celecoxib-PVP K30-Ryoto sugar ester L1695; and (F) celecoxib-PVP K30-D-α-tocopheryl

polyethylene glycol 1000 succinate (K30-TPGS).

Molecules 2014, 19 20329

Table 1. Formulation, particle size, and specific surface area of celecoxib-

polyvinylpyrrolidone (celecoxib-PVP) solid dispersion nanoparticles prepared using the

supercritical antisolvent (SAS) process.

Formulation (Weight) Drug Content

(%) # Mean Particle

Size (nm) Specific Surface

Area (m2/g)

Celecoxib:PVP K30 = 4:6 90.6 ± 2.3 158.3 ± 19.8 78.1 ± 1.3 Celecoxib:PVP K30 = 3:7 92.7 ± 3.1 150.1 ± 15.5 80.4 ± 1.4 Celecoxib:PVP K30 = 2:8 94.5 ± 1.9 155.3 ± 12.1 81.2 ± 1.8

Celecoxib:PVP K30:Gelucire 44/14 = 2:5:3 96.9 ± 1.1 610.3 ± 71.2 21.2 ± 1.2 Celecoxib:PVP K30:Poloxamer 188 = 2:5:3 95.3 ± 1.0 275.1 ± 33.4 40.1 ± 1.1 Celecoxib:PVP K30:Poloxamer 407 = 2:5:3 94.5 ± 1.3 282.7 ± 40.5 36.8 ± 1.1

Celecoxib:PVP K30:Ryoto sugar ester L1695 = 2:5:3 95.8 ± 2.4 220.1 ± 23.4 49.7 ± 1.3 Celecoxib:PVP K30:TPGS = 2:5:3 95.1 ± 1.8 290.8 ± 44.7 29.2 ± 1.0

# The celecoxib concentration within the solid dispersion nanoparticles was determined by dissolving about

30 mg of solid dispersion nanoparticles in 100 mL ethanol, filtering aliquots using a 0.45-μm syringe filter, and

analyzing concentration with an HPLC system The drug content (%) = weight of the drug in nanoparticles/weight

of the feeding excipients and drug × 100. Data are expressed as the mean ± standard deviation (n = 3).

Figure 2. The particle size distribution of celecoxib-PVP K30 solid dispersion nanoparticles

prepared using the SAS process.

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Figure 3. Differential scanning calorimetry thermograms (A) and powder X-ray diffraction

patterns (B) of celecoxib-PVP K30 solid dispersion nanoparticles prepared using the SAS

process. Cel: celecoxib.

In the dissolution studies (Figure 4), raw celecoxib showed very low dissolution because of its poor

water solubility. However, the dissolution of celecoxib was significantly increased by solid dispersion

nanoparticles. The dissolution of drug from celecoxib-PVP (2:8) solid dispersion nanoparticles had a

maximum rate of 33.0% within 0.25 h, and gradually decreased to 26.3% after 2 h (Figure 4). The

maximum dissolution and dissolution percentage of celecoxib at 2 h, increased with increasing PVP

ratio within nanoparticles, but there were no significant differences between PVP solid dispersion

nanoparticles (4:6, 3:7, and 2:8). The enhanced dissolution properties of celecoxib might be due to the

formation of amorphous celecoxib in PVP solid dispersion nanoparticles prepared by the SAS process

and the specific interaction of PVP and celecoxib in forming hydrogen bonds [25]. It has also been

previously reported that recrystallization of celecoxib from supersaturated solution was significantly

inhibited by PVP in a concentration-dependent manner [24,25]. Gupta et al. reported that the solubility

of amorphous celecoxib was 20.43 μg/mL in PVP aqueous solution and 4.57 μg/mL in water, indicating

that PVP enhances solubilization and stabilization of amorphous celecoxib [26]. The dissolution of

celecoxib was also enhanced by incorporated of surfactant within the solid dispersion nanoparticles. In

particular, the maximum celecoxib dissolution in celecoxib-PVP K30-TPGS solid dispersion nanoparticles

was 64%, and dissolution at 2 h was 56%. The dissolution percentage of celecoxib at 2 h ranked by the

SNK test ranged as follows: celecoxib-PVP K30-poloxamer 188 < celecoxib-PVP K30-Ryoto sugar ester

L1695 < celecoxib-PVP K30-poloxamer 407 < celecoxib-PVP K30-gelucire 44/14 < celecoxib-PVP

K30-TPGS solid dispersion nanoparticle. In solubility studies, the most effective surfactant tested was

TPGS, followed by gelucire 44/14 and poloxamer 407, as shown in Table 2. Dissolution of celecoxib

from solid dispersion nanoparticles with surfactant at 2 h was in fact related to the solubilization

capability of the surfactant used. As reported in our previous study, the solubility of celecoxib was

significantly increased by TPGS via micellar solubilization at a critical micelle concentration of

0.1 mg/mL [6]. Furthermore, it was reported that the recrystallization of celecoxib from amorphous

state can be effectively inhibited by the combination of PVP and TPGS [27]. Therefore, the significant

enhancement in both the dissolution rate and extent of dissolution of celecoxib can probably be

Molecules 2014, 19 20331

attributed to the increased solubility induced by the PVP-TPGS stabilized amorphous form, the

enhanced solubility by micellar solubilization of TPGS, and/or the improved wettability of particle

surface by PVP and surfactant. In addition, celecoxib-PVP K30-TPGS solid dispersion nanoparticles

showed higher dissolution above 60% at different dissolution media pH (1.2, 4.0, 6.8 and water), as

shown in Figure 5. Interestingly, the dissolution of celecoxib from celecoxib-PVP K30-TPGS solid

dispersion nanoparticles was not influenced by the pH of dissolution media. The solubility of celecoxib

was not influenced by the physiological pH condition (1.0–7.5) since the pKa of celecoxib is 11.1. In

fact, raw celecoxib showed very low dissolution of below 3% in different pH dissolution media (pH

1.2, 4.0, 6.8 and water). To determine the IVIVC for celecoxib, the dissolution efficiency (DE%) as

defined by Khan and Rhodes, was calculated using the dissolution profiles of celecoxib.

Figure 4. Dissolution profiles of celecoxib-PVP K30 solid dispersion nanoparticles

prepared using the SAS process in pH 1.2 dissolution medium. Data are expressed as the

mean ± standard deviation (n = 3).

Table 2. Solubility of celecoxib in various surfactant solutions.

Surfactant Solubility (μg/mL) #

Raw celecoxib 3.9 ± 0.2 Gelucire 44/14 3100.1 ± 65.8 Poloxamer 188 14.8 ± 3.2 Poloxamer 407 2060.7 ± 40.3

Ryoto Sugar Ester L1695 1380.9 ± 54.2 TPGS 6522.3 ± 25.0

# The solubility of celecoxib was measured in 1% surfactant solution at 37 °C. Data are expressed as the

mean ± standard deviation (n = 3).

Molecules 2014, 19 20332

Figure 5. Effect of dissolution media on the dissolution of celecoxib-PVP K30-TPGS

solid dispersion nanoparticles prepared using the SAS process. Data are expressed as the

mean ± standard deviation (n = 3).

The dissolution efficiency for celecoxib-PVP solid dispersion nanoparticles was calculated from the

area under the dissolution curves at 120 min and expressed as a percentage of the area of the rectangle

resulting from 100% dissolution within the same period [28]. As shown in Table 3, celecoxib-PVP

K30-TPGS solid dispersion nanoparticles showed the highest DE%.

Table 3. Dissolution efficiency of celecoxib-PVP solid dispersion nanoparticles prepared

using the SAS process.

Formulation DE (%) #

Raw celecoxib 1.9 ± 0.2 Celecoxib:PVP K30 = 2:8 31.1 ± 2.6

Celecoxib:PVP K30: Gelucire 44/14 = 2:5:3 43.8 ± 3.0 Celecoxib:PVP K30:Poloxamer 188 = 2:5:3 23.6 ± 2.0 Celecoxib:PVP K30:Poloxamer 407 = 2:5:3 37.9 ± 2.8

Celecoxib:PVP K30:Ryoto Sugar Ester L1695 = 2:5:3 29.3 ± 3.1

Celecoxib:PVP K30:TPGS = 2:5:3 59.1 ± 3.3 # Data are expressed as the mean ± standard deviation (n = 3).

The bioavailability of celecoxib solid dispersion nanoparticles and raw celecoxib was determined in

SD rats. As shown in the plasma concentration–time curves (Figure 6), the Cmax of celecoxib from solid

dispersion nanoparticles was significantly increased compared to raw celecoxib. From the pharmacokinetic

data, Cmax and AUC0→24 h of raw celecoxib was 1.14 μg/mL and 14.42 μg·h/mL, respectively (Table 4).

As expected from the dissolution data, celecoxib-PVP K30-TPGS solid dispersion nanoparticles showed

highest Cmax and AUC0→24 h values and were 4.6 and 5.7 times higher, respectively, that shown by raw

celecoxib. Furthermore, ANOVA showed that there were significant differences among the samples

Molecules 2014, 19 20333

(p < 0.05), which in order of increasing the Cmax of celecoxib, were ranked by the SNK test as follows:

raw celecoxib < celecoxib-PVP K30 (2:8) < celecoxib-PVP K30-poloxamer 407 < celecoxib-PVP

K30-TPGS solid dispersion nanoparticle. Further studies of correlation between the in vitro dissolution

data and in vivo pharmacokinetic parameters (Figure 7) showed in vitro DE was well correlated to

in vivo Cmax, and AUC0→24 h (R2 > 0.90). In fact, in vitro dissolution of celecoxib was increased by

solid dispersion nanoparticles, resulting in increased oral bioavailability. This also implies that the oral

bioavailability of celecoxib can be controlled by the in vitro dissolution property. This correlation was

similar to previously reported IVIVC studies for celecoxib using SMEDDS formulation and silica-lipid

hybrid formulation [4,5]. As reported in previous studies, IVIVC study using DE is an effective method

for analyzing celecoxib in solid dispersion nanoparticles [18].

Figure 6. Plasma concentration-time profile of celecoxib in rats after oral administration of

the raw celecoxib and celecoxib-PVP K30 solid dispersion nanoparticles prepared using

the SAS process. Data are expressed as the mean ± standard deviation (n = 5).

Table 4. Pharmacokinetic parameters of celecoxib-PVP solid dispersion nanoparticles

prepared using the SAS process.

Formulation AUC0→24 h (μg·h/mL) Cmax (μg/mL) Tmax (h)

Raw celecoxib 14.42 ± 5.59 1.14 ± 0.63 4.0 ± 1.2Celecoxib:PVP K30 = 2:8 31.63 ± 10.38 a 2.82 ± 0.92 a 3.6 ± 1.5

Celecoxib:PVP K30:Poloxamer 407 = 2:5:3 47.39 ± 12.56 ab 4.52.6 ± 1.12 ab 2.0 ± 1.0Celecoxib:PVP K30:TPGS = 2:5:3 65.78 ± 15.56 a–c 6.49 ± 1.53 a–c 1.4 ± 1.1

a indicates p < 0.05 vs. raw celecoxib; b indicates p < 0.05 vs. Celecoxib:PVP K30; c indicates p < 0.05 vs.

Celecoxib:PVP K30:Poloxamer 407. Data are expressed as the mean ± standard deviation (n = 5).

Molecules 2014, 19 20334

Figure 7. Correlation between the in vitro dissolution efficiency and in vivo pharmacokinetic

parameters of celecoxib. (A) Area under the curve (AUC) and (B) Cmax.

Generally, supersaturatable formulations such as solid dispersions, raise the highly supersaturated

state of poorly water-soluble APIs above their equilibrium solubility in in vitro dissolution medium

and in vivo in the gastrointestinal tract [29]. To enhance the bioavailability of poorly water-soluble

APIs by a supersaturatable system, the formulation must have the essential properties of generation

and maintenance of the thermodynamically metastable supersaturated state [30]. Therefore, the

precipitation of APIs must be inhibited by using a hydrophilic polymer and surfactant. In this study,

the enhanced solubility of celecoxib induced by celecoxib-PVP-TPGS solid dispersion nanoparticles

resulted in enhanced oral absorption through the gastrointestinal epithelial membrane. This formulation

can also be applied to potentially enhance the bioavailability of other poorly water-soluble APIs.

3. Experimental Section

3.1. Materials

Celecoxib was obtained from Dong-A ST (Yongin, Korea). Gelucire 44/14 (melting point, 44 °C,

Gattefossè, Saint-Priest, France), polyvinylpyrrolidone (PVP K30, BASF Co. Ltd., Ludwigshafen,

Germany), poloxamer 188 (melting point: 52 °C, BASF Co. Ltd., Ludwigshafen, Germany),

poloxamer 407 (melting point: 56 °C BASF Co. Ltd., Ludwigshafen, Germany), Ryoto sugar ester

L1695 (melting point: 35–50 °C, Mitsubishi-Kagaku Foods Co., Tokyo, Japan), and D-α-tocopheryl

polyethylene glycol 1000 succinate (melting point: 37 °C, TPGS, Eastman Co., Kingsport, TN, USA)

were used. Hydrochlorothiazide was purchased from Sigma-Aldrich Co. Ltd (St. Louis, MO, USA).

Acetonitrile, ethanol, and methanol were of high-performance liquid chromatography (HPLC) grade.

3.2. Preparation of Celecoxib-PVP K30 Solid Dispersion Nanoparticles

Celecoxib solid dispersion nanoparticles were manufactured by the SAS process using Thar

SAS200 equipment (Thar Technologies, Pittsburgh, PA, USA) [31,32]. To study the effect of the

celecoxib/PVP K30 ratio, celecoxib-PVP K30 nanoparticles were fabricated with 20%, 30%, and 40%

drug loading concentrations. The drug solution was first prepared by dissolving celecoxib and PVP

K30 in methanol at 5% solute concentration. Once the particle precipitation vessel reached steady state

Molecules 2014, 19 20335

(above critical temperature and pressure), the drug solution was introduced into the particle

precipitation vessel by an HPLC liquid pump. The SAS process was then performed under the

following conditions, based on preliminary experiments [15]: temperature of precipitation vessel,

40 °C; pressure of precipitation vessel, 15 MPa; flow rate of drug solution, 1 mL/min; and flow rate of

supercritical carbon dioxide, 11 g/min. To completely extract residual methanol, supercritical carbon

dioxide was introduced into the precipitation vessel for 1 h, and the celecoxib solid dispersion

nanoparticles were obtained from the precipitation vessel after depressurization to atmospheric

pressure level. To investigate the effect of various surfactants on the dissolution and bioavailability of

celecoxib, ternary solid dispersion nanoparticles of celecoxib-PVP K30 and surfactants were also

prepared. Drug solutions were prepared by dissolving celecoxib, PVP K30, and the respective

surfactant (with the exception of gelucire 44/14) in methanol at 5% solute concentration. For gelucire

44/14, the drug solution was prepared using a 1:1 mixture of methanol and dichloromethane. Gelucire

44/14, poloxamer 188, poloxamer 407, Ryoto sugar ester L1695, and TPGS were tested as surfactants.

The nanoparticles were manufactured under the same conditions as described above for the

formulation without surfactant. The formulation characteristics of celecoxib solid dispersion

nanoparticles are presented in Table 1.

3.3. Characterization of Celecoxib-PVP K30 Solid Dispersion Nanoparticles

The morphology of celecoxib-PVP K30 solid dispersion nanoparticles was examined using a

scanning electron microscope (SEM, JSM-6300, Jeol Ltd., Tokyo, Japan). After suspension of the

nanoparticles in mineral oil by sonication for 20 min, the particle size of celecoxib-PVP K30 solid

dispersion nanoparticles was determined by the dynamic light scattering method using a laser particle

analyzer (BI-9000; Brookhaven, NY, USA). The specific surface area (m2/g) of celecoxib-PVP solid

dispersion nanoparticles was measured by the Brunauer, Emmett, and Teller (BET) method with

nitrogen as the adsorption gas using an Autosorb-1 instrument (Quantachrome GmbH, Odelzhausen,

Germany). The crystalline state of celecoxib within the solid dispersion nanoparticle was characterized

using a differential scanning calorimeter (DSC, S-650 model, Sinco Co. Ltd., Seoul, Korea) and powder

X-ray diffractometer (PXRD, D8 Advance X-ray diffraction system, Bruker AXS GmbH, Karlsruhe,

Germany). DSC was calibrated for temperature and enthalpy using indium. The melting temperature

and enthalpy of celecoxib and solid dispersion nanoparticles (2–5 mg) were then recorded at a heating

rate of 5°/min under nitrogen purge (20 mL/min). Powder X-ray diffraction pattern was recorded from

5° to 50° of 2θ at a scanning rate of 3°/min. The celecoxib concentration within the solid dispersion

nanoparticles was determined by dissolving about 30 mg of solid dispersion nanoparticles in 100 mL

ethanol, filtering aliquots using a 0.45-μm syringe filter, and analyzing concentration with an HPLC

system (Agilent 1100 Series, Agilent Technologies, Santa Clara, CA, USA). Chromatographic separation

was performed with a CAPCELL PAK C18 UG120 (4.6 mm × 150 mm, 5 μm) reversed-phase column

(Shiseido Fine Chemicals, Tokyo, Japan). Acetonitrile in water (60%) was used as the isocratic mobile

phase at a flow rate of 1.0 mL/min. Celecoxib was detected by an ultraviolet (UV) detector at 238 nm.

The drug content was calculated using the following equation: weight of the drug in nanoparticles/weight

of the feeding excipients and drug × 100. Dissolution tests for raw celecoxib and celecoxib-PVP K30

solid dispersion nanoparticles were performed in 300 mL of a dissolution medium containing

Molecules 2014, 19 20336

hydrochloric acid (HCl) and sodium chloride (NaCl) at pH 1.2 and 37 °C using a USP rotating paddle

apparatus at 50 rpm (non-sink condition). Samples equivalent to 60 mg of celecoxib were added to the

dissolution tester (Electrolab, Mumbai, India). At predetermined time intervals, 2 mL of the medium

was sampled, filtered using a 0.45-μm glass fiber syringe filter, diluted with methanol, and analyzed

for celecoxib concentration by HPLC as described above. Dissolution tests were also performed using

acetate buffer (pH 4.0), phosphate buffer (pH 6.8) and water under the same conditions.

3.4. Solubility of Celecoxib in Surfactant Solution

To investigate the solubilization capability of gelucire 44/14, poloxamer 188, poloxamer 407, Ryoto

sugar ester L1695, and TPGS, which were tested as surfactants, the solubility of celecoxib was

measured in 1% surfactant solution at 37 °C. About 50 mg celecoxib was added to 5 mL of surfactant

solution in glass vials. After sonication for 1 h, vials were placed in a shaking water bath at 100 rpm

for 3 days. After setting solution aside for 4 h, 3 mL aliquots were filtered using a 0.45-μm syringe filter,

diluted with methanol and then celecoxib concentration was analyzed by HPLC as described above.

3.5. Pharmacokinetic Study in Rats

The study protocol complied with the institutional guidelines for the care and use of laboratory

animals, and it was approved by the ethics committee of Kyungsung University (No. 2014-04A). Oral

bioavailability of celecoxib-PVP solid dispersion nanoparticles was evaluated in fasted Sprague-Dawley

(SD) rats weighing 250 ± 10 g. Twenty SD rats were divided in to four groups (n = 5), and the first

group was orally administered raw celecoxib, while the remaining three groups received solid dispersion

nanoparticles of celecoxib-PVP K30, celecoxib-PVP K30-poloxamer 407, or celecoxib-PVP K30-TPGS,

using an animal feeding needle at a dose of 100 mg/kg of celecoxib. Prior to oral dosing, the samples

were dispersed in distilled water. Following a predetermined time interval, about 0.35 mL of blood

was drawn from the retro-orbital plexus of the rats, collected in heparinized tubes, and centrifuged at

10,000 rpm for 5 min at 4 °C to obtain the plasma. The plasma concentration of celecoxibwas

subsequently measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Sample

preparation and conditions for analysis adhered to previously reported methods [3–5]. For protein

precipitation, 20 μL of a 20 μg/mL solution of hydrochlorothiazide (internal standard; IS) was added to

100 μL of heparinized plasma followed by 400 μL of acetonitrile. After vortexing briefly, the organic

phase was separated from the aqueous phase by centrifugation at 13,000 rpm for 5 min. A 5-μL aliquot

of the organic phase was injected into the LC-MS/MS system. Chromatographic separation was

achieved using a ZORBAX® Eclipse XDB-C 18 column (4.6 mm × 50 mm, 1.8 μm, Agilent

Technologies). The HPLC system was operated isocratically at 40 °C. The mobile phase consisted of

0.1% formic acid:acetonitrile (25:75, v/v). The mass spectrometer (Agilent technologies 6410 triple

quadrupole mass spectrometer) was equipped with an electrospray source. The ions monitored using

multiple reaction monitoring were m/z 380 (parent) and m/z 316 (product) for celecoxib, and m/z 296

(parent), and m/z 205 (product) for the IS. Pharmacokinetic analysis of the data was carried out with

WinNonlin Standard Edition software, Version 5.3 (Pharsight Corp., St. Louis, MO, USA). The area

under the curve (AUC0→24 h) was calculated using the trapezoidal method. The maximum

Molecules 2014, 19 20337

concentration of celecoxib after oral administration (Cmax) and the time to reach the maximum

concentration (Tmax) were determined from the experimentally obtained data.

3.6. Statistical Analysis

The data were analyzed by a one-way analysis of variance (ANOVA) test followed by the

Student-Newman-Keuls (SNK) and least-squares difference (LSD) tests using the SPSS 21.0 software

(IBM SPSS Statistics, Armonk, NY, USA).

4. Conclusions

In this study, spherical celecoxib solid dispersion nanoparticles smaller than 300 nm were

successfully developed using the SAS process. Among the formulations studied, celecoxib-PVP-TPGS

solid dispersion nanoparticles showed significantly enhanced in vitro dissolution and oral absorption of

celecoxib. In addition, in vitro dissolution efficiency was well correlated to in vivo pharmacokinetic

parameters (Cmax, and AUC). The present study therefore demonstrated that the formulation of

celecoxib-PVP-TPGS solid dispersion nanoparticles using the SAS process is a highly effective

strategy for enhancing the bioavailability of poorly water-soluble celecoxib.

Acknowledgments

This research was supported by Basic Science Research Program through the National Research

Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning

(NRF-2014R1A1A1006492).

Author Contributions

The listed authors contributed to this work as described in the following: Eun-Sol Ha and

Gwang-Ho Choo carried out the experiment and prepared the manuscript. Min-Soo Kim (corresponding

author) and In-Hwan Baek conducted the experimental design and revised the manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

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Sample Availability: Not available.

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